Curr Atheroscler Rep23:71 )5 02( DOI 10.1007/s11883-015-0510-0

VASCULAR BIOLOGY (T HLA, SECTION EDITOR)

Potential Contributions of Intimal and Plaque Hypoxia to Atherosclerosis Guo-Hua Fong 1

# Springer Science+Business Media New York 2015

Abstract Injury of arterial endothelium by abnormal shear stress and other insults induces migration and proliferation of vascular smooth muscle cells (VSMCs), which in turn leads to intimal thickening, hypoxia, and vasa vasorum angiogenesis. The resultant new blood vessels extend from the tunica media into the outer intima, allowing blood-borne oxidized low-density lipoprotein (oxLDL) particles to accumulate in outer intimal tissues by extravasation through local capillaries. In response to oxLDL accumulation, monocytes infiltrate into arterial wall tissues, where they differentiate into macrophages and subsequently evolve into foam cells by uptaking large quantities of oxLDL particles, the latter process being stimulated by hypoxia. Increased oxygen demand due to expanding macrophage and foam cell populations contributes to persistent hypoxia in plaque lesions, whereas hypoxia further promotes plaque growth by stimulating angiogenesis, monocyte infiltration, and oxLDL uptake into macrophages. Molecularly, the accumulation of hypoxia-inducible factor (HIF)-1α and the expression of its target genes mediate many of the hypoxia-induced processes during plaque initiation and growth. It is hoped that further understanding of the underlying mechanisms may lead to novel therapies for effective intervention of atherosclerosis. Keywords Hypoxia . Angiogenesis . Atherosclerosis . Inflammation . Vasa vasorum . Intima This article is part of the Topical Collection on Vascular Biology * Guo-Hua Fong [email protected] 1

Center for Vascular Biology and Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA

Introduction According to the model originally proposed by Russell Ross [1, 2], atherosclerosis is the result of a catastrophic chain of events beginning with injuries to arterial endothelium, which mostly occur at arterial bifurcations due to local shear stress. Endothelial cell (EC) injuries are worsened by multiple pathological conditions including hypertension, dyslipidemia, hyperglycemia, and chronic infection [3, 4]. Following endothelial injury, vascular smooth muscle cells (VSMCs) are activated to undergo proliferation and migration, resulting in intimal thickening. Accompanying these events are the extravasation and intimal deposition of oxidized low-density lipoprotein (oxLDL). Furthermore, injured ECs express leukocyte adhesion molecules and chemokines, which mediate the recruitment of leukocytes including mostly monocytes and to a lesser extent T cells. Once recruited into intimal tissues, monocytes differentiate into macrophages and engulf large amounts of oxLDL to become foam cells, which are giant cells filled with lipid droplets [5]. The swelling number of foam cells and macrophages comprises the majority of cells in an atherosclerotic plaque. Other cellular constituents of a plaque lesion include VSMCs, T cells, B cells, dendritic cells, fibroblasts, erythrocytes, bone marrow-derived mononuclear cells, and endothelial cells. The expansion of plaque tissue mass leads to local hypoxia stress due to increases in both oxygen consumption and intercapillary distances. In response to tissue hypoxia, the vasa vasorum vasculature is activated to undergo angiogenesis [6]. While angiogenesis supports further plaque growth, the new blood vessels are limited to the base and shoulder areas of a plaque lesion, leaving avascular core areas where large numbers of foam cells undergo apoptotic death. At advanced stages, atherosclerotic lesions are fragile protrusions filled with lipid droplets, leaky and unstable blood vessels, loosely

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distributed cells (mostly foam cells), disintegrated extracellular matrix, and a thin fibrous cap on the arterial luminal side [6–9]. When the fibrous cap fails to resist hemodynamic stress, plaque rupture occurs, causing severe hemorrhage and thrombosis due to the collapse of intraplaque blood vessels. Depending on the location of plaque rupture events, blockage of arterial blood flow by the resultant thrombi may lead to myocardial infarction, stroke, or peripheral artery diseases. While the currently prevailing model accommodates much of the data in the field of atherosclerosis, there are still some unexplained facts. For example, although intimal thickening is typically cited as an early event, its specific relevance to specific processes such as leukocyte infiltration remains vague. Furthermore, while oxLDL is thought to accumulate in the intima by extravasation through injured arterial endothelium, in reality, oxLDL accumulation occurs in intimal locations too far to reach by direct diffusion through the inner intimal tissues [10••]. Recent advances in the field of hypoxia regulation of atherosclerosis provide new insights into potential mechanisms of atherosclerosis. Over the last several years, various authors have reviewed specific issues related to hypoxia regulation of atherosclerosis [6–9, 11–13]. The intention of this review

Fig. 1 Atherosclerosis is self-perpetuating due to positive feedback between plaque growth and hypoxia stress. The process of atherosclerosis is arbitrarily divided into ten discrete steps for illustrative purpose, although the process is continuous. The intima in human coronary arteries is composed of two layers of structures: the inner intima containing scattered VSMCs and the outer intima consisting of multiple layers of VSMCs [10••]. Under normal conditions, both the inner and outer intimal tissues are avascular. Various events starting from EC injury until the formation of a large

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article is to complement the existing literature and provide an integrative view on how hypoxia dynamically interacts with a range of processes including the initiation and expansion of plaque lesions, angiogenesis, lipid accumulation, and inflammation (Fig. 1 and Table 1). Therapeutic potential of the hypoxia signaling pathway in preventing and treating atherosclerosis is discussed as well.

oxLDL Deposition in the Outer Intimal Tissues Is Not Explained by Entry Through Arterial Endothelium The extravasation of oxLDL into the arterial wall is a key event in atherosclerosis, which begins prior to the formation of visible plaque lesions but continues throughout the entire course of plaque development. The widely popularized theory of atherosclerosis presumes that oxLDL particles leak out of the circulation through injured arterial endothelium, where they initiate atherosclerotic plaque development by attracting the infiltration of inflammatory cells such as monocytes and T cells [31]. If oxLDL extravasation indeed occurs through the arterial endothelium, one would expect its accumulation to occur first in the inner intima just underneath the endothelium.

plaque lesion are indicated with color-coded arrows. Blue arrows indicate progression from earlier arterial wall events (such as EC injury) to subsequent changes, gray arrows indicate events that contribute to hypoxia stress, purple arrows indicate hypoxia-dependent events, and thick black arrow at the last step indicates the formation of a large and necrotic plaque lesion through collaboration between hypoxia, angiogenesis, leukocyte infiltration, and oxLDL uptake. Although not included, plaque lesions may also contain other leukocytes such as T cells, B cells, and dendritic cells

Some events are both contributors to hypoxia and induced or promoted by hypoxia. For example, leukocyte infiltration is promoted by hypoxia for a number of reasons, such as hypoxia-induced expression of proinflammatory cytokines. However, infiltration of leukocytes especially monocytes leads to hypoxia as a result of increased metabolic burden. Likewise, oxLDL deposition promotes leukocyte infiltration which thus contributes to hypoxia; however, oxLDL deposition is also facilitated by hypoxia-induced angiogenesis

[25•, 26–30]

[14, 21–24]

[14–16] [17–20]

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VSMC proliferation and migration Increased oxygen consumption oxLDL deposition; monocyte infiltration; foam Expression and secretion of angiogenic factors cell formation (VEGF-A, etc.) and proinflammatory cytokines; oxLDL uptake Expansion Leading to hypoxia Expansion of plaque lesion; formation and Leukocyte infiltration and retention; VSMC Oxygen consumption; oxLDL deposition; expression expansion of necrotic core proliferation and migration; macrophage of proinflammatory cytokines and macrophage and foam cell survival retention factors Promoted by hypoxia Angiogenesis; expansion of plaque lesion; Leukocyte infiltration; and retention; formation oxLDL deposition; HIF-1α accumulation; expression formation and expansion of necrotic core of more foam cells; apoptosis of angiogenic factors, proinflammatory cytokines, (in more severely hypoxic areas) and MMPs; glycolysis (lactate accumulation); ROS production; degradation of extracellular matrix

Cellular level Morphological level Nature of events

Leading to hypoxia Intimal thickening Promoted by hypoxia Angiogenesis; formation of fatty streaks Initiation

Table 1

Hypoxia during initiation and expansion stages of atherosclerotic plaque development

Molecular level

Refs

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However, the earliest oxLDL deposition is detected deep in the musculoelastic layer of the tunica intima (outer intima), rather than in the inner intima [32, 33]. While one could argue that oxLDL particles might quickly pass through the inner intima and directly deposit in the outer intimal tissues, there are strong counterarguments against this scenario [10••]. First, if oxLDL particles indeed enter the arterial wall through the arterial endothelium, they would have to diffuse through the inner intima before reaching the outer intima. Since this is presumed to be an ongoing process accompanying atherosclerotic plaque growth, the diffusion process should leave at least a trail of oxLDL particles in the inner intima. However, oxLDL is detectable in the outer intima only [10••]. Second, VSMCs are abundantly present between the site of oxLDL accumulation and arterial endothelium [10••]. Since the average diameter of oxLDL particles is approximately 20 nm, it would be highly counterintuitive to assume that these large particles could efficiently diffuse through muscular intimal tissues. Overall, these structural features seem to argue against the theory that oxLDL particles enter the outer intima directly through the injured arterial endothelium.

oxLDL and Monocytes May Enter the Outer Intima Directly Through the Vasa Vasorum Vasculature Oxygenation by diffusion is efficient only within the distance of several cell layers. Thus, vascular wall tissues of large arteries are oxygenated by the vasa vasorum vasculature. In normal human coronary arteries, vascularization by the vasa vasorum is limited to the adventitia and medial layers, whereas the intima including its outer layer is generally avascular. Because the avascular outer intima is fairly distant from the arterial lumen, it is reasonable to assume that this tissue layer may be hypoxic, especially when its distance to the arterial lumen is further increased by intimal thickening [10••, 34]. Hypoxia-induced expression of angiogenic factors may trigger the vasa vasorum to generate new branches to vascularize the outer intima and atherosclerotic plaque lesions. There are data from both human donors and animal models indicating that vasa vasorum angiogenesis may occur very early in atherosclerosis, presumably in response to hypoxia associated with intimal thickening. In human carotid arteries, microvessels were found in very early plaque lesions [17]. In human aortic segments with early stage plaques, intramedial vascular density was increased and microvessels grew towards the outer intima [18]. In a swine model of high cholesterol diet, inhibition of angiogenesis with thalidomide strongly inhibited the early stage development of atherosclerotic plaques [19]. Because outer intima thickening eventually progresses into vascularized atherosclerotic lesions, it can be inferred that angiogenesis is an ongoing process throughout plaque lesion

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development. Since angiogenesis should alleviate tissue hypoxia, it may be curious why hypoxia persists and angiogenesis continues. The precise mechanism remains unknown, but some rational speculations are possible. Intimal thickening itself can be induced by endothelial injuries on the arterial luminal side, which is independent of angiogenesis. In addition, angiogenesis may provide the local VSMCs with nutrition and oxygen and thus accelerates intimal thickening. Continuing intimal thickening and subsequent plaque growth may cancel out the oxygenation effect of newly formed blood vessels, thus maintaining a chronically hypoxic environment to induce further angiogenesis. The base of a vasa vasorum vascular network is directly connected to the same artery whose wall tissues it supplies. Thus, arterial blood constituents may readily divert into vasa vasorum microvessels, including those extended into the outer intima in response to intimal thickening and hypoxia. Furthermore, since hypoxia promotes vascular permeability by inducing robust expression of VEGF-A [35], oxLDL particles may extravasate into the hypoxic outer intima through leaky microvessels. Likewise, monocytes and other leukocytes may enter the outer intima through the vasa vasorum. In support of such a mechanism, in early atherosclerotic plaques of human carotid arteries, capillary-like microvessels were found to be associated with macrophages, agreeing with the above analysis [17]. In contrast to the above model, the currently prevailing theory states that oxLDL leaks into the intima through the injured arterial endothelium, which displays increased vascular permeability in part due to disruption of cadherin-based junctions. Further mechanistic studies are needed to evaluate the relative contributions of each of these two mechanisms.

that 60–70 % of statin-treated individuals continue on to develop atherosclerosis [37]. The above controversies can be better understood under the hypothesis that oxLDL extravasation may occur mostly through microvessels that invade the outer intima [10••]. According to this hypothesis, vasa vasorum angiogenesis into the outer intima is a prerequisite to oxLDL deposition in arterial wall tissues. Once the outer intima becomes vascularized, oxLDL may leak out of the vascular system and deposit into outer intimal tissues, even in individuals with normal levels of plasma LDL concentration. Nonetheless, plasma LDL concentration may affect the extent and pace of oxLDL accumulation, thus modulating the rate of atherosclerosis.

The Hypoxia Signaling Pathway Contributes to the Initiation and Growth of Atherosclerotic Plaque Lesions Consistent with a role of hypoxia in atherosclerosis, the presence of arterial wall hypoxia has been demonstrated. In LDLR-deficient mice, hypoxia was detected in aortic arterial wall tissues as early as in the plaque initiation stage [25•]. However, it remains to be clarified whether hypoxia was mostly confined to the outer intimal layer. In ApoE−/− mice maintained on normal chow, atherosclerotic plaques formed only after chronic exposure to hypoxia (3 weeks) [26]. When ApoE−/− mice were fed with high cholesterol diet, plaques formed under both hypoxia and normoxia, but exposure to hypoxia accelerated plaque growth. By contrast, oxygenation of ApoE−/− mice with carbogen inhibited the growth of atherosclerotic plaques [38]. These studies suggest that at least systemic hypoxia may indeed contribute to atherosclerosis, but further studies are needed to assess the specific role of hypoxia signaling in the outer intima. A major mediator of hypoxia-induced effects in atherosclerosis is hypoxia-inducible factor-1α (HIF-1α) [21], which is a transcription factor activating the expression of a large set of hypoxia-induced genes, including those encoding proteins important for angiogenesis, metabolism, erythropoiesis, and cell survival. As summarized in Table 2 and further explained below, HIF-1α abundance is predominantly regulated at the level of protein stability, but to some extent at the mRNA level as well [45, 46]. A list of putative HIF-1α target genes involved in atherosclerotic plaque development was tabulated

Outer Intima Angiogenesis May Be Causal to Atherosclerosis A widely popularized concept in atherosclerosis is that high levels of plasma LDL may lead to atherosclerosis. But this statement is inaccurate. Individuals with normal plasma LDL levels account for as much as approximately 50 % of hospitalized coronary heart disease patients [36]. Furthermore, while it is commonly agreed that lowering LDL-cholesterol levels with HMG-CoA reductase inhibitors (statins) is the gold standard therapy to prevent atherosclerosis, the fact is Table 2 Plaque-borne triggers of HIF-1α expression and accumulation

Triggers

Mechanism

Tests

Supporting data

Refs

ROS oxLDL Hypoxia asTF

PHD suppression ROS production PHD suppression Unknown

In vitro In vitro In plaque lesions In plaque lesions

Increased HIF-1α protein Increased HIF-1α protein Increased HIF-1α protein Increased HIF-1α mRNA expression

[39•, 40, 41] [39•, 42] [21, 23, 43] [44]

HIF-1α, HIF-2α Hypoxia Hypoxia HIF-1α HIF-1α

Hypoxia HIF-1α CD40L, Ang II, HIF-1α Macrophages Macrophages Mononuclear cells, endothelial cells; VSMCs

Macrophages Macrophages Macrophages Macrophages Atherosclerotic arteries

Unc5b Ntn1 Mif1α/MIF

Versican Pro-IL-1ß Sema3E Lox1 HIG2

Roles Cell types or tissues

Vasa vasorum angiogenesis may begin at the initiation stage and continue throughout the duration of plaque growth [6, 12, 13, 20, 21, 45, 62, 63]. Angiogenesis mostly invades the base and shoulder areas of expanding plaque lesions, providing nearby plaque tissues with oxygen and nutrients. Importantly, vascularization by vasa vasorum vasculature also provides routes for circulating monocytes and other leukocytes to infiltrate into plaque tissues. Blood vessels vascularizing atherosclerotic plaques are typically devoid of mural cells such as pericytes and VSMCs,

Genes/Proteins

Hypoxia May Stimulate Plaque Growth by Activating Vasa Vasorum Angiogenesis

Table 3

Atherosclerotic plaques are strongly hypoxic, especially in the necrotic core [13, 28]. Plaque hypoxia can be detected by two techniques, including (1) labeling with hypoxia markers such as pimonidazole or 18F-labeled EF5 (2-(2-nitro-1H-imidazol-1-yl)N-(2,2,3,3,3-pentafluoropropyl) acetamide), and (2) uptake of 18 F-fluorodeoxyglucose (18F-FDG) into macrophages. The accumulation of 18F-FDG is likely substantial in plaque lesions, where a large number of hypoxic macrophages consume large quantities of glucose due to robust glycolysis for ATP production [57–60]. In animal models, hypoxia markers are detected by immunohistochemical staining or autoradiography. In human patients, sensitive and high-resolution detection of 18F signals is accomplished by a combination of positron-emission tomography (PET) and computed tomography (CT) [59, 61].

Hypoxia-induced expression in atherosclerotic plaques (identified since 2009)

Plaque Hypoxia Can Be Detected by Labeling with Hypoxia Markers or PET-CT Imaging

Macrophage retention Macrophage retention and survival Inhibits macrophage migration; plaque instability; promotes VSMC proliferation and migration Retention of lipoproteins Proinflammatory Macrophage retention oxLDL uptake into macrophages Lipid accumulation

Mediated by

Refs

by Sluimer JC and Daemen MJ in 2009 [45]. Since then, additional hypoxia-regulated genes have been linked to atherosclerosis [27, 47–51, 52•, 53], which are summarized in Table 3. In normal tissues, prolyl hydroxylase domain-containing proteins (PHDs; including PHD1, PHD2, and PHD3) are mostly responsible for initiating HIF-α protein degradation [43, 54, 55]. PHDs are Fe + + and α-ketoglutarate-dependent dioxygenases which employ O2 as a substrate to hydroxylate HIF-α proteins at specific prolyl residues, labeling them for E3 ligase-dependent polyubiquitination and proteasomal degradation [43]. Under hypoxia, HIF-α proteins accumulate to high levels due to diminished PHD activities. Besides hypoxia, other mechanisms also exist which suppress PHD activities. For example, reactive oxygen species (ROS) oxidize Fe++ to Fe+++, thus inhibiting PHD activities by depleting an essential cofactor [56]. The production of ROS is stimulated by different mechanisms during inflammation, such as oxidative stress- and oxLDL-mediated signaling [14, 22, 23, 39•, 42, 57].

[49] [50] [51] [52•] [53]

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and display incomplete endothelial cell-cell junctions [64]. Such vascular structures are highly permeable and prone to destructions. The precise mechanisms underlying vascular abnormalities in plaque lesions have not been delineated. However, elevated VEGF-A expression may be at least partially responsible for deficiencies in mural cell recruitment [65]. In agreement with important roles of angiogenesis in plaque growth, experimental stimulation of vasa vasorum angiogenesis promoted plaque growth [66], whereas inhibition of angiogenesis had opposite effects [67–71]. Similarly, local overexpression of dominant-negative HIF-α in injured arterial wall tissues inhibited the production of angiogenic proteins (VEGF-A, Flk-1, and Flt-1) and suppressed plaque lesion growth [71], whereas overexpression of functional HIF-1α or HIF-2α further increased plaque sizes [71].

Hypoxia May Promote Plaque Growth by Stimulating Inflammation An important component of atherosclerotic plaque growth is the recruitment of leukocytes especially monocytes, and their differentiation first into macrophages and subsequently into foam cells. The extravasation of oxLDL particles and their uptake by macrophages and foam cells provide much of the substance that feeds into plaque growth. These processes are selfperpetuating through mutual augmentation between hypoxia and inflammation. Although macrophages and foam cells rely on glycolysis for ATP production under hypoxic conditions, if oxygen is available, they do carry out oxidative phosphorylation. Thus, the expansion of the macrophage and foam cell populations is accompanied by increased oxygen demand for as long as oxygen is available to them, until the tissue microenvironment becomes sufficiently hypoxic to force these cells into glycolysis. In turn, hypoxia promotes monocyte recruitment and oxLDL uptake by macrophages, leading to a vicious positive feedback cycle between plaque expansion and hypoxia. Hypoxia may promote inflammation by different mechanisms [29]. Hypoxia or HIF-1α promotes the expression of many proinflammatory cytokines which may mediate the recruitment of monocytes and other leukocytes into plaque lesions. Examples of such cytokines include stromal differentiation factor-1 (SDF-1; also called CXCL12), tumor necrosis factor-α (TNF-α), interleukin-1β, VEGF-A, and monocyte chemoattractant protein-1 (MCP-1) [30, 50, 72–75]. Hypoxia or HIF-1α also induces the expression of several macrophage retention molecules, including netrin-1, Unc5b, and semaphorin 3E (the last of which is also upregulated by oxLDL) [27, 51], which facilitate inflammation by retaining macrophages in plaque tissues. Hypoxia activates the expression of scavenger receptor lectin-like oxLDL receptor (Lox-1), which mediates oxLDL uptake into macrophages [52•]. Hypoxia-dependent oxLDL

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uptake into macrophages leads to the expansion of the foam cell population, which constitute the majority of the cells in atherosclerotic plaques. When foam cells die, oxLDL particles are released during their defragmentation. oxLDL activates the expression of proinflammatory cytokines by a variety of cells such as macrophages, VSMCs, etc., thus promoting the recruitment of more monocytes into plaque lesions [76, 77].

Hypoxia May Contribute to Plaque Instability by Multiple Mechanisms Many of the events induced or facilitated by hypoxia contribute to the instability of atherosclerotic plaque lesions. For example, hypoxia-stimulated monocyte infiltration and oxLDL uptake together lead to the accumulation of foam cells, which are fragile due to the storage of large quantities of lipid droplets. Vasa vasorum angiogenesis, which is also hypoxia dependent, contributes to plaque instability in several different ways. Besides providing routes for oxLDL extravasation and monocyte recruitment which eventually lead to more foam cells, poor vascular integrity also allows erythrocytes to escape from the circulation and into plaque tissues. Following their arrival at plaque tissues, erythrocytes break down due to apoptotic cell death, releasing large amounts of cholesterol [78]. These events increase lipid contents in atherosclerotic plaques, thus contributing to plaque instability [79]. Hypoxia promotes macrophage survival, thus favoring the expansion of macrophage and foam cell populations [80]. Because foam cells are loaded with lipids, increased number of foam cells translates into increased instability. In the avascular core where the tissue environment is extremely hypoxic, ATP depletion occurs, resulting in significant foam cell death and formation of a large necrotic core filled with lipids [81]. In addition, hypoxia activates the expression and secretion of metalloproteases by a variety of cells such as macrophages, which contribute to the weakening of plaque structures by degrading extracellular matrix proteins [7, 26].

Clinical Potential of the Hypoxia Pathway for the Prevention and Treatment of Atherosclerosis The ability of hypoxia to regulate various aspects of atherosclerosis raises the possibility that the hypoxia signaling pathway may be a promising target for therapeutic intervention. Although there have been no reported clinical trials directly based on the hypoxia signaling pathway, studies in animal models have provided encouraging results. For example, local inhibition of HIF signaling by adenoviral vector-mediated expression of HIF dominant-negative mutants strongly suppressed atherosclerotic plaque development in ApoE−/− mice [71]. Plaque angiogenesis and growth were also inhibited by

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anti-angiogenic inhibitors sFlt-1 (sVEGFR-1) and endostatin [62, 82, 83]. Applications of several small molecule antiangiogenic inhibitors also effectively inhibited vasa vasorum angiogenesis, leukocyte infiltration, and plaque growth. These include TNP-470, thalidomide, and saponins from Panax notoginseng [19, 62, 83, 84]. Interestingly, vaccination against angiogenic receptors Tie-2 or VEGFR-2 was effective in inhibiting plaque angiogenesis and plaque growth [85, 86].

Concluding Remarks and Future Directions While targeting the hypoxia pathway and vasa vasorum angiogenesis which may have great therapeutic potential, several important issues remain to be addressed. First, early intervention will be critical. Targeting angiogenesis too late in the process could trigger the rupture of fragile plaque lesions and lead to devastating consequences. Second, partial suppression of angiogenesis could exacerbate plaque hypoxia by disrupting intraplaque blood supply, which could potentially lead to unintended outcomes due to multiple roles of hypoxia in promoting plaque growth. Third, the hypoxia signaling pathway is important for many different physiological processes, and many angiogenic molecules are also important for vascular endothelial cell survival in various tissues. Thus, systemic targeting of hypoxia signaling or angiogenesis could result in undesirable side effects. To address these issues, it will be highly desirable to understand local events in early plaque lesions that influence hypoxia, angiogenesis, and inflammation. Future studies will be most beneficial by keeping in mind the need of specifically suppressing hypoxia signaling and angiogenesis in early plaque lesions.

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12. Compliance with Ethics Guidelines Conflict of Interest G-H Fong declares no conflicts of interest.

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Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

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